《英语教学》（英文版）8 RNA Purification

Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-471 (Electronic) 8 RNA Purification Lori A. Martin, Tiffany. Smith, Dawn Obermoeller Brian Bruner Martin Kracklauer and Subramanian Dharmaraj Selecting a Purification Strategy Do Your Experiments Require Total RNa or mRNA? 198 Is It possible to predict the total rna yield from a Certain mass of tissue or number of cells? 20 Is There Protein in Your RNA Preparation, and If So, Should You be concerned 202 Is Your RNa Physically Intact? Does It Matter? Which Total RNA Isolation Technique Is Most Appropriate for Your research? What Protocol modifications should Be used for RNA Isolation from difficult tissues? Is a One-Step or Two-Step mRNA(poly (A)RNA)- Purification Strategy Most Appropriate for Your Situation? 209 How Many Rounds of Oligo(dT)-Cellulose Purification Are required? 210 Which Oligo(dT)-Cellulose Format Is Most 210 Can Oligo(dT)-Cellulose Be Regenerated and Reused?.. 211 Can a Kit Designed to Isolate mRNA Directly from he Biological Sample Purify mRNA from Total RNA?... 212 Maximizing the Yield and Quality of an RNA Preparation.. 212 What Constitutes“ RNase- Free Technique”?

197 8 RNA Purification Lori A. Martin, Tiffany J. Smith, Dawn Obermoeller, Brian Bruner, Martin Kracklauer, and Subramanian Dharmaraj Selecting a Purification Strategy . . . . . . . . . . . . . . . .. . . . . . . . . 198 Do Your Experiments Require Total RNA or mRNA? . . . . . 198 Is It Possible to Predict the Total RNA Yield from a Certain Mass of Tissue or Number of Cells? . . . . . . . . 201 Is There Protein in Your RNA Preparation, and If So, Should You Be Concerned? . . . . . . . . . . . . . . . . . . . . 202 Is Your RNA Physically Intact? Does It Matter? . . . . . . . . . . 202 Which Total RNA Isolation Technique Is Most Appropriate for Your Research? . . . . . . . . . . . . . . . . . . . . . 203 What Protocol Modifications Should Be Used for RNA Isolation from Difficult Tissues? . . . . . . . . . . . . . . . . 207 Is a One-Step or Two-Step mRNA-(poly(A) RNA)- Purification Strategy Most Appropriate for Your Situation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 How Many Rounds of Oligo(dT)–Cellulose Purification Are Required? . . . . . . . . . . . . . . . . . . . . . . . . . 210 Which Oligo(dT)–Cellulose Format Is Most Appropriate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 Can Oligo(dT)–Cellulose Be Regenerated and Reused? . . . 211 Can a Kit Designed to Isolate mRNA Directly from the Biological Sample Purify mRNA from Total RNA? . . . 212 Maximizing the Yield and Quality of an RNA Preparation . . . 212 What Constitutes “RNase-Free Technique”? . . . . . . . . . . . . 212 Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)

How Does depc inhibit rnase? 2|3 How Are DEPC-Treated Solutions Prepared? Is or Should You Treat Your Pre-made Reagents with r213 More depc better Should You Prepare Reagents with DEPC-Treated Wate DEPC? ..2|4 How Do You Minimize RNA Degradation during Sample Collection and storage ..2|4 How Do You Minimize RNA Degradation during Sample Disruption? Is There a Safe Place to Pause during an rna Purification Procedure? 2|8 What Are the Options to Quantitate Dilute RNA 2|8 What Are the Options for Storage of Purified RNAS 2l9 Troubleshooting 220 A Pellet of Precipitation RNA Is Not Seen at the End of he rna Purification A Pellet Was Generated, but the Spectrophotometer Reported a Lower Reading Than Expected, or Zero Absorbance RNA Was Prepared in Large Quantity, but in a downstream Reaction RT PCr is Example My Total RNA Appeared as a Smear in an Ethidum Bromide-stained Denaturing Agarose Gel; 18S and 28S RNA Bands Were not observed 222 Only a Fraction of the Original RNA Stored at-70C Remained after Storage for Six Months 222 Bibliography SELECTING A PURIFICATION STRATEGY Do Your Experiments Require Total RNA or mRNA? One of the first decisions that the researcher has to make when detecting or quantitating RNA is whether to isolate total RNA or poly (A)-selected RNa (also commonly referred to as mRNA) This choice is further complicated by the bewildering array of RNA isolation kits available in the marketplace. In addition the downstream application influences this choice. The following section is a short primer in helping make that decision From a purely application point of view, total RNA might suffice for most applications, and it is frequently the starting material for applications ranging from the detection of an mRNA species by Northern hybridization to quantitation of a message by Martin et al

How Does DEPC Inhibit RNase? . . . . . . . . . . . . . . . . . . . . . . 213 How Are DEPC-Treated Solutions Prepared? Is More DEPC Better? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Should You Prepare Reagents with DEPC-Treated Water, or Should You Treat Your Pre-made Reagents with DEPC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 How Do You Minimize RNA Degradation during Sample Collection and Storage? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 How Do You Minimize RNA Degradation during Sample Disruption? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Is There a Safe Place to Pause during an RNA Purification Procedure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 What Are the Options to Quantitate Dilute RNA Solutions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 What Are the Options for Storage of Purified RNA? . . . . . . . 219 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 A Pellet of Precipitation RNA Is Not Seen at the End of the RNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 A Pellet Was Generated, but the Spectrophotometer Reported a Lower Reading Than Expected, or Zero Absorbance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 RNA Was Prepared in Large Quantity, but it Failed in a Downstream Reaction: RT PCR is an Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 My Total RNA Appeared as a Smear in an Ethidum Bromide-stained Denaturing Agarose Gel; 18S and 28S RNA Bands Were not Observed . . . . . . . . . . . . . . . . 222 Only a Fraction of the Original RNA Stored at -70°C Remained after Storage for Six Months . . . . . . . . . . . . . . 222 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 SELECTING A PURIFICATION STRATEGY Do Your Experiments Require Total RNA or mRNA? One of the first decisions that the researcher has to make when detecting or quantitating RNA is whether to isolate total RNA or poly(A)-selected RNA (also commonly referred to as mRNA). This choice is further complicated by the bewildering array of RNA isolation kits available in the marketplace. In addition the downstream application influences this choice. The following section is a short primer in helping make that decision. From a purely application point of view, total RNA might suffice for most applications, and it is frequently the starting material for applications ranging from the detection of an mRNA species by Northern hybridization to quantitation of a message by 198 Martin et al

RT-PCR. The preference for total RNA reflects the challenge of purifying enough poly (A) RNA for the application (mRNA comprises <5% of cellular RNA), the potential loss of a particu- lar message species during poly(A)purification, and the difficulty uantitating small amounts of purified poly (A)RNA. If the data generated with total Rna do not meet your expectations, using poly() RNa instead might provide the sensitivity and pecificity that your application requires. The pros and cons with either choice are discussed below. Your experimental data will provide the best guidance in deciding whether to use total or poly(A)RNA. Be flexible and open minded; there are many vari- ables to consider when making this decision Two situations where using poly (A)RNA is essential are cDNA library construction, and preparation of labeled CDNA for hybridization to gene arrays. To avoid generating cDNA libraries with large numbers of ribosomal clones, and nonspecific labeled cDNA it is crucial to start with poly(A)RNA for these procedures. The next section gives a brief description of the merits and demerits of using total RNA or poly (A)RNA in some of the most common RNA analysis techniques. Chapter 14, "Nucleic Acid Hybridization, discusses the nuances and quirks of these proce dures in greater depth. For detailed RNa purification protocol see Krieg(1996)Rapley and Manning(1998), and Farrel (1998) Northern Hybridizations Northern analysis is the only technique available that can deter mine the molecular weight of an mRNA species. It is also the least sensitive. Total RNA is most commonly used in this assay, but if you dont detect the desired signal, or if false positive signals from ribosomal RNA are a problem, switching to poly(A)RNA might be a good idea. Since only very small amounts of poly(A)RNA are present, make sure that it is feasible and practical to obtain enough starting cells or tissue. Theoretically you could use as much as 30 ug of poly(A)RNA in a Northern, which is the amount found in approximately 1 mg of total RNA. Will it be practical and feasible for you to sacrifice the cells or tissue required to get this much RNA? If not, use as much poly(A)RNA as is practical One drawback to using poly (A)RNA in Northern hybridiza tions is the absence of the ribosomal rna bands, which are ord of the rna amples, as discussed later in this chapter. Fortunately there are other strategies besides switching to poly(A)RNa that can be used to increase the sensitivity of Northern hybridizations. You could alter the hybridization conditions of the Dna probe RNA Purification 199

RT-PCR. The preference for total RNA reflects the challenge of purifying enough poly(A) RNA for the application (mRNA comprises <5% of cellular RNA), the potential loss of a particu￾lar message species during poly(A) purification, and the difficulty in quantitating small amounts of purified poly(A) RNA. If the data generated with total RNA do not meet your expectations, using poly(A) RNA instead might provide the sensitivity and specificity that your application requires. The pros and cons with either choice are discussed below. Your experimental data will provide the best guidance in deciding whether to use total or poly(A) RNA. Be flexible and open minded; there are many vari￾ables to consider when making this decision. Two situations where using poly(A) RNA is essential are cDNA library construction, and preparation of labeled cDNA for hybridization to gene arrays. To avoid generating cDNA libraries with large numbers of ribosomal clones, and nonspecific labeled cDNA it is crucial to start with poly(A) RNA for these procedures. The next section gives a brief description of the merits and demerits of using total RNA or poly(A) RNA in some of the most common RNA analysis techniques. Chapter 14, “Nucleic Acid Hybridization,” discusses the nuances and quirks of these proce￾dures in greater depth. For detailed RNA purification protocols, see Krieg (1996) Rapley and Manning (1998), and Farrel (1998). Northern Hybridizations Northern analysis is the only technique available that can deter￾mine the molecular weight of an mRNA species. It is also the least sensitive. Total RNA is most commonly used in this assay, but if you don’t detect the desired signal, or if false positive signals from ribosomal RNA are a problem, switching to poly(A) RNA might be a good idea. Since only very small amounts of poly(A) RNA are present, make sure that it is feasible and practical to obtain enough starting cells or tissue.Theoretically you could use as much as 30mg of poly(A) RNA in a Northern, which is the amount found in approximately 1 mg of total RNA. Will it be practical and feasible for you to sacrifice the cells or tissue required to get this much RNA? If not, use as much poly(A) RNA as is practical. One drawback to using poly(A) RNA in Northern hybridiza￾tions is the absence of the ribosomal RNA bands, which are ordi￾narily used to gauge the quality and relative quantity of the RNA samples, as discussed later in this chapter. Fortunately there are other strategies besides switching to poly(A) RNA that can be used to increase the sensitivity of Northern hybridizations. You could alter the hybridization conditions of the DNA probe RNA Purification 199

Anderson, 1999), or you could switch to using RNA probes in the hybridization, which are 3- to 5-fold more sensitive than DNA probes in typical hybridization buffers (Ambion Technical Bulletin 168, and references therein). Dramatic differences in the sensitivity of northern blots can also be seen from using different hybridization buffers. If you remain dissatisfied with the Northern data, and you are not interested in determining the size of the target, switching to a more sensitive technique such as nuclease protection or RT-PCR might help. Nuclease protection assays, which are 5-to 10-fold more sensitive than traditional membrane hybridizations, can accommodate 80 to 100 ug of nucleic acid in a single experiment RT-PCR can detect extremely rare messages, for example, 400 copies of a message in a 1 ug sample as described by Sun et al ( 1998). RT-PCR is currently the most sensitive of the RNa analy- sis techniques, enabling detection and quantitation of the rarest of targets. Quantitative approaches have become increasingly reliable with introduction of internal standards such as in com- petitive PCR strategies(Totzke et aL., 1996; Riedy et al., 1995) Dot slot blots In this procedure, RNA samples are directly applied to a mem- brane, either manually or under vacuum through a filtration manifold. Hybridization of probe to serial dilutions of sample can quickly generate quantitative data about the expression level of a target. Total RNA or poly (A)RNA can be used in this assay. Since the RNa is not size-fractionated on an agarose gel, a potential drawback to using total RNA in dot/slot blots is that signal of interest cannot be distinguished from cross-hybridization t rRNA. Switching to poly(A)RNA as the target source might alle- viate this problem. However. it is crucial that relevant positive and negative controls are run with every dot/slot blot, whether the source of target nucleic acid is total RNA or poly(A)RNA Hybridization to Gene Arrays and Reverse Dot blots Gene arrays consist of cDNA clones(sometimes in the form of PCR products, sometimes as oligonucleotides)or the correspond- ing oligos spotted at high density on a nylon membrane, glass slide, or other solid support. By hybridizing labeled cDNA probes reverse transcribed from mRNA, the expression of potentially hundreds of genes can be simultaneously analyzed. This procedure requires that the labeled cDNa be present in excess of the target spotted on the array. This is difficult to achieve unless poly(a) RNA is used as template in the labeling reaction 200 Martin et al

(Anderson, 1999), or you could switch to using RNA probes in the hybridization, which are 3- to 5-fold more sensitive than DNA probes in typical hybridization buffers (Ambion Technical Bulletin 168, and references therein). Dramatic differences in the sensitivity of Northern blots can also be seen from using different hybridization buffers. If you remain dissatisfied with the Northern data, and you are not interested in determining the size of the target, switching to a more sensitive technique such as nuclease protection or RT-PCR might help. Nuclease protection assays, which are 5- to 10-fold more sensitive than traditional membrane hybridizations, can accommodate 80 to 100mg of nucleic acid in a single experiment. RT-PCR can detect extremely rare messages, for example, 400 copies of a message in a 1mg sample as described by Sun et al. (1998). RT-PCR is currently the most sensitive of the RNA analy￾sis techniques, enabling detection and quantitation of the rarest of targets. Quantitative approaches have become increasingly reliable with introduction of internal standards such as in com￾petitive PCR strategies (Totzke et al., 1996; Riedy et al., 1995). Dot/Slot Blots In this procedure, RNA samples are directly applied to a mem￾brane, either manually or under vacuum through a filtration manifold. Hybridization of probe to serial dilutions of sample can quickly generate quantitative data about the expression level of a target.Total RNA or poly(A) RNA can be used in this assay. Since the RNA is not size-fractionated on an agarose gel, a potential drawback to using total RNA in dot/slot blots is that signal of interest cannot be distinguished from cross-hybridization to rRNA. Switching to poly(A) RNA as the target source might alle￾viate this problem. However, it is crucial that relevant positive and negative controls are run with every dot/slot blot, whether the source of target nucleic acid is total RNA or poly(A) RNA. Hybridization to Gene Arrays and Reverse Dot Blots Gene arrays consist of cDNA clones (sometimes in the form of PCR products, sometimes as oligonucleotides) or the correspond￾ing oligos spotted at high density on a nylon membrane, glass slide, or other solid support. By hybridizing labeled cDNA probes reverse transcribed from mRNA, the expression of potentially hundreds of genes can be simultaneously analyzed.This procedure requires that the labeled cDNA be present in excess of the target spotted on the array. This is difficult to achieve unless poly(A) RNA is used as template in the labeling reaction. 200 Martin et al

Ribonuclease Protection Assays Either total RNA or poly(A)RNA can be used as starting mate rial in nuclease protection assays. However, total RNa usually af- fords enough sensitivity to detect even rare messages, when the maximum amount(as much as 80 to 100 ug)is used in the assay If the gene is expressed at extremely low levels, requiring week long exposure times for detection, a switch to poly (A)RNA might prove beneficial and may justify the added cost. Although very sensitive, nuclease protection assays do require laborious gel purification of the full-length probe to avoid getting confusing RT-PCR RT-PCR is the most sensitive method for detecting and quant tating mRNA. Theoretically, even very low-abundance messages can be detected with this technique. Total RNA is routinely used as the template for RT-PCR, (Frohman, 1990) but some cloning situations and rare messages require the use of poly(A)RNA (Amersham Pharmacia Biotech, 1995) Note that one school of thought concerning RT-PCR considers it advisable to treat the sample rna with DNase I, since no purifi cation method produces RNA completely free of contaminating genomic DNA. RT-PCR is sensitive enough that even very small amounts of genomic DNA contamination can cause false posi tives. A second school of thought preaches avoidance of DNase I as discussed in Chapter 11, " PCR cDNA Library Synthesis As mentioned earlier, high-quality mRNA that is essentially free of ribosomal RNA is required for constructing CDNA libra ries. Unacceptably high backgrounds of ribosomal RNa clones would be produced if total RNa were reverse transcribed to pre pare CDNA Is It possible to predict the total rna yield from a certain Mass of tissue or number of cells? The data provided in this section are based on experimentation at Ambion, Inc. using a variety of samples and different purifica tion products. The reader is cautioned that these are theoretical estimates, and yields can vary widely based on the type of tissue or cells used for the isolation, especially when dealing with difficult samples, as discussed later. The importance of rapid and complete tissue disruption, and homogenizing at subfreezing tem RNA Purification

Ribonuclease Protection Assays Either total RNA or poly(A) RNA can be used as starting mate￾rial in nuclease protection assays. However, total RNA usually af￾fords enough sensitivity to detect even rare messages, when the maximum amount (as much as 80 to 100mg) is used in the assay. If the gene is expressed at extremely low levels, requiring week￾long exposure times for detection, a switch to poly(A) RNA might prove beneficial and may justify the added cost. Although very sensitive, nuclease protection assays do require laborious gel purification of the full-length probe to avoid getting confusing results. RT-PCR RT-PCR is the most sensitive method for detecting and quanti￾tating mRNA. Theoretically, even very low-abundance messages can be detected with this technique. Total RNA is routinely used as the template for RT-PCR, (Frohman, 1990) but some cloning situations and rare messages require the use of poly(A) RNA (Amersham Pharmacia Biotech, 1995). Note that one school of thought concerning RT-PCR considers it advisable to treat the sample RNA with DNase I, since no purifi- cation method produces RNA completely free of contaminating genomic DNA. RT-PCR is sensitive enough that even very small amounts of genomic DNA contamination can cause false posi￾tives. A second school of thought preaches avoidance of DNase I, as discussed in Chapter 11, “PCR.” cDNA Library Synthesis As mentioned earlier, high-quality mRNA that is essentially free of ribosomal RNA is required for constructing cDNA libra￾ries. Unacceptably high backgrounds of ribosomal RNA clones would be produced if total RNA were reverse transcribed to pre￾pare cDNA. Is It Possible to Predict the Total RNA Yield from a Certain Mass of Tissue or Number of Cells? The data provided in this section are based on experimentation at Ambion, Inc. using a variety of samples and different purifica￾tion products. The reader is cautioned that these are theoretical estimates, and yields can vary widely based on the type of tissue or cells used for the isolation, especially when dealing with difficult samples, as discussed later. The importance of rapid and complete tissue disruption, and homogenizing at subfreezing tem￾RNA Purification 201

peratures cannot be overemphasized. In addition, yields from very small amounts of starting material are subject to the law of dimin ishing returns. Thus, if the option is available, always choose more starting material rather than less Samples can be pooled together, if possible, to maximize yields. For example 5 mg of tissue or 2.5 x 10 cells yields about 10 of total RNA, comprised of 8ug rRNA, 0.3 ug mRNA, 1.7 ug tRNA, and other RNA. In comparison, 1 g of tissue or 5 x 10 cells yields about 2mg of total RNA, comprised of 1.6mg rRNA 60ug mRNA +333 ug tRNA and other rna Is There Protein in Your RNA Preparation, and If So, Should You be concerned? Pure RNA has an A260: A20 absorbance ratio of 2.0. However for most applications, a low A260: A2so ratio probably wont affect the results, Researchers at ambion Inc have used total rna with A260 2s0 ratios ranging from 1. 4 to 1. 8 with good results in RNase protection assays, Northern analysis, in vitro translation experi ments, and RT-PCR assays. If protein contamination is suspe to be causing problems, additional organic extractions with an equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1 mixture)may remove the contaminant Residual phenol can also lower the A260: A2so ratio, and inhibit downstream enzymatic reac tions Chloroform/isoamyl alcohol (24: 1)extraction will remove residual phenol. Chapter 4, " How to Properly Use And Maintain Laboratory Equipment, discusses other artifacts that raise and lower the A2602s0 ratio Some tissues will consistently produce RnA with a lower A260-2s0 ratio than others the A260-2s0 ratio for rna iso- lated from liver and kidney tissue, for example, is rarely above 1.7. Is Your RNa Physically Intact? Does It Matter? The integrity of your rNa is best determined by electrophore sis on a formaldehyde agarose gel under denaturing conditions. The samples can be visualized by adding 10 ug/ml of Ethidium Bromide(EtBr)(final concentration) to the sample before load ng on the gel. Compare your prep's 28S rRNa band (located at approximately 5 Kb in most mammalian cells) to the 18S rRNA band (located at approximately 2.0 Kb in most mammalian cells In high-quality rna the 28S band should be approximately twice the intensity of the 18S band(Figure 8.1). The most sensitive test of Rna integrity is northern analy sing a high molecular weight probe expressed at low levels in the sues being analyzed. However, this method of quality control is very time-consuming and is not necessary in most cases 202 Martin et al

peratures cannot be overemphasized. In addition, yields from very small amounts of starting material are subject to the law of dimin￾ishing returns. Thus, if the option is available, always choose more starting material rather than less. Samples can be pooled together, if possible, to maximize yields. For example, 5 mg of tissue or 2.5 ¥ 106 cells yields about 10mg of total RNA, comprised of 8mg rRNA, 0.3mg mRNA, 1.7mg tRNA, and other RNA. In comparison, 1 g of tissue or 5 ¥ 108 cells yields about 2 mg of total RNA, comprised of 1.6mg rRNA + 60mg mRNA + 333mg tRNA and other RNA. Is There Protein in Your RNA Preparation, and If So, Should You Be Concerned? Pure RNA has an A260 :A280 absorbance ratio of 2.0. However, for most applications, a low A260 :A280 ratio probably won’t affect the results. Researchers at Ambion, Inc. have used total RNA with A260:280 ratios ranging from 1.4 to 1.8 with good results in RNase protection assays, Northern analysis, in vitro translation experi￾ments, and RT-PCR assays. If protein contamination is suspected to be causing problems, additional organic extractions with an equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1 mixture) may remove the contaminant. Residual phenol can also lower the A260 :A280 ratio, and inhibit downstream enzymatic reac￾tions. Chloroform/isoamyl alcohol (24:1) extraction will remove residual phenol. Chapter 4, “How to Properly Use And Maintain Laboratory Equipment,” discusses other artifacts that raise and lower the A260:280 ratio. Some tissues will consistently produce RNA with a lower A260:280 ratio than others; the A260:280 ratio for RNA iso￾lated from liver and kidney tissue, for example, is rarely above 1.7. Is Your RNA Physically Intact? Does It Matter? The integrity of your RNA is best determined by electrophore￾sis on a formaldehyde agarose gel under denaturing conditions. The samples can be visualized by adding 10mg/ml of Ethidium Bromide (EtBr) (final concentration) to the sample before load￾ing on the gel. Compare your prep’s 28S rRNA band (located at approximately 5Kb in most mammalian cells) to the 18S rRNA band (located at approximately 2.0Kb in most mammalian cells). In high-quality RNA the 28S band should be approximately twice the intensity of the 18S band (Figure 8.1). The most sensitive test of RNA integrity is Northern analysis using a high molecular weight probe expressed at low levels in the tissues being analyzed. However, this method of quality control is very time-consuming and is not necessary in most cases. 202 Martin et al

1234567891011 Figure 8.1 Assessing qual- ity of rNa preparation via 9.5 (A) This gel shows total RNA samples (5ug/lane) ranging from high-quality, intact RNa (lane 2)to almost 4.4 totally degraded RNA (lane 7). Note that as the rNA is 2.4 degraded, the 28S and 18S ribosomal bands become less distinct, the intensity of the 1.35- ribosomal bands relative to the background staining in the lane is reduced. and there is a significant shift in their apparent size as compared size standards. (B)This autorad of the same gel hybridization with a biotinylated GAPDH RNA probe followed by noniso- topic detection. The exposur is 10 minutes the day after the chemiluminescent sub. strate was applied. Note that the signal in lane 2, from intact RNA. is well local- 点二三 ing below the bands, or when the RNa is extremely de- graded, no bands at all (lane f Ambion. In Northern analysis is not tolerant of partially degraded RNA samples are even slightly degraded, the quality of the data is severely compromised. For example, even a single cleavage in 20% of the target molecules will decrease the signal on a North ern blot by 20%. Nuclease protection assays and RT-PCR analy ses will tolerate partially degraded RNA without compromising the quantitative nature of the results. Which Total RNA Isolation Technique Is Most Appropriate for Your research? There are three basic s &y n a chaotropic agent such as guani- thods of isolating total RNA from cells and tissue samples. mos dium or a detergent to break open the cells and simultaneously RNA Purification 203

Northern analysis is not tolerant of partially degraded RNA. If samples are even slightly degraded, the quality of the data is severely compromised. For example, even a single cleavage in 20% of the target molecules will decrease the signal on a North￾ern blot by 20%. Nuclease protection assays and RT-PCR analy￾ses will tolerate partially degraded RNA without compromising the quantitative nature of the results. Which Total RNA Isolation Technique Is Most Appropriate for Your Research? There are three basic methods of isolating total RNA from cells and tissue samples. Most rely on a chaotropic agent such as guani￾dium or a detergent to break open the cells and simultaneously RNA Purification 203 9.5 – 7.5 – 4.4 – 2.4 – 1.35 – .24 – 1 2 3 4 5 6 7 8 9 10 11 Figure 8.1 Assessing qual￾ity of RNA preparation via agarose gel electrophoresis (A) This gel shows total RNA samples (5mg/lane) ranging from high-quality, intact RNA (lane 2) to almost totally degraded RNA (lane 7). Note that as the RNA is degraded, the 28S and 18S ribosomal bands become less distinct, the intensity of the ribosomal bands relative to the background staining in the lane is reduced, and there is a significant shift in their apparent size as compared to the size standards. (B) This is an autorad of the same gel after hybridization with a biotinylated GAPDH RNA probe followed by noniso￾topic detection. The exposure is 10 minutes the day after the chemiluminescent sub￾strate was applied. Note that the signal in lane 2, from intact RNA, is well local￾ized with minimal smearing, whereas the signals from degraded RNA samples show progressively more smear￾ing below the bands, or when the RNA is extremely de￾graded, no bands at all (lane 7). Reprinted by permission of Ambion, Inc. A B

inactivate RNases. The lysate is then processed in one of several ways to purify the RNa away from protein, genomic DNA, and other cellular components. a brief description of each method along with the time and effort involved, the quality of rna obtained, and the scalability of the procedures follow Guanidium-Cesium Chloride Method Slow, laborious procedure, but RNA is squeaky clean; unsuitable for large sample numbers; little if any genomic DNA remains. This method employs guanidium isothiocyanate to lyse cells and simultaneously inactivate ribonucleases rapidly. The cellular RNA is purified from the lysate via ultracentrifugation through a cesium chloride or cesium trifluoroacetate cushion since rna is more tube after 12 to 24 hours of centrifugation at 232, 000rpul? Of th dense than DNA and most proteins, it pellets at the bott This classic method yields the highest-quality RNA of any avail- able technique. Small RNAs(e. g, 5S RNA and tRNAs) cannot be prepared by this method as they will not be recovered(Mehra 1996). The original procedures were time-consuming, laborious, and required overnight centrifugation. The number and size of amples that could be processed simultaneously were limited by the number of spaces in the rotor. Commercial products have been developed to replace this lengthy centrifugation (Paladichuk, 1999)with easier, less time-consuming methods. However, if the goal were to isolate very high-quality RNA from a limited number of samples, this would be the method of choice (Glisin, Crkuenjakov and Byus, 1974) Single- and Multiple Step Guanidium Acid-Phenol Method Faster, fewer steps, prone to genomic DNA contamination, some what cumbersome if large sample numbers are to be processed The guanidium-acid phenol procedure has largely replaced the cesium cushion method because RNA can be isolated from a large number of samples in two to four hours(although somewhat cum bersome) without resorting to ultracentrifugation. RNA mole cules of all sizes are purified, and the technique can be easily scaled up or down to process different sample sizes. The single step method( Chomczynski and Sacchi, 1987) is based on the propensity of RNa molecules to remain dissolved in the aqueous phase in a solution containing 4 M guanidium thiocyanate, pH 4.0, in the presence of a phenol/chloroform organic phase. At this low pH, DNA molecules remain in the organic phase, whereas proteins and other cellular macromolecules are retained at the Martin et al

inactivate RNases. The lysate is then processed in one of several ways to purify the RNA away from protein, genomic DNA, and other cellular components. A brief description of each method along with the time and effort involved, the quality of RNA obtained, and the scalability of the procedures follow. Guanidium-Cesium Chloride Method Slow, laborious procedure, but RNA is squeaky clean; unsuitable for large sample numbers; little if any genomic DNA remains. This method employs guanidium isothiocyanate to lyse cells and simultaneously inactivate ribonucleases rapidly.The cellular RNA is purified from the lysate via ultracentrifugation through a cesium chloride or cesium trifluoroacetate cushion. Since RNA is more dense than DNA and most proteins, it pellets at the bottom of the tube after 12 to 24 hours of centrifugation at ≥32,000rpm. This classic method yields the highest-quality RNA of any avail￾able technique. Small RNAs (e.g., 5S RNA and tRNAs) cannot be prepared by this method as they will not be recovered (Mehra, 1996). The original procedures were time-consuming, laborious, and required overnight centrifugation. The number and size of samples that could be processed simultaneously were limited by the number of spaces in the rotor. Commercial products have been developed to replace this lengthy centrifugation (Paladichuk, 1999) with easier, less time-consuming methods. However, if the goal were to isolate very high-quality RNA from a limited number of samples, this would be the method of choice (Glisin, Crkuenjakov and Byus, 1974). Single- and Multiple Step Guanidium Acid-Phenol Method Faster, fewer steps, prone to genomic DNA contamination, some￾what cumbersome if large sample numbers are to be processed. The guanidium-acid phenol procedure has largely replaced the cesium cushion method because RNA can be isolated from a large number of samples in two to four hours (although somewhat cum￾bersome) without resorting to ultracentrifugation. RNA mole￾cules of all sizes are purified, and the technique can be easily scaled up or down to process different sample sizes. The single￾step method (Chomczynski and Sacchi, 1987) is based on the propensity of RNA molecules to remain dissolved in the aqueous phase in a solution containing 4M guanidium thiocyanate, pH 4.0, in the presence of a phenol/chloroform organic phase. At this low pH, DNA molecules remain in the organic phase, whereas proteins and other cellular macromolecules are retained at the interphase. 204 Martin et al

It is not difficult to find researchers who swear by GITC- phenol procedures because good-quality RNA, free from geno- mic dNa contamination is quickly produced. However, a se- cond camp of researchers avoid these same procedures because they often contain contaminating genomic DNA (Lewis, 1997 S. Herzer, personal communication). There is no single expla nation for these polarized opinions, but the following should be considered Problems can occur in the procedure during the phenol/chloro form extraction step. The mixture must be spun with sufficient force to ensure adequate separation of the organic and aqueous layers; this will depend on the rotor as can be seen in Table 8.1 For best results the centrifuge brake should not be applied, nor should it be applied to gentler settings The interface between the aqueous and organic layers is another potential source of genomic contamination. To get higl purity RNA, avoid the white interface(can also appear cream colored or brownish) between the two layers; leave some of the aqueous layer with the organic layer. If RNA yield is crucial, you'll probably want as much of the aqueous layer as possible e. again leaving the white interface. In either case you can repeat the organic extraction until no white interface is seen. Residual salt from the precipitation step, appearing as a huge white pellet, can interfere with subsequent reactions. Excessiv salt should be suspected when a very large white pellet is obtained from an RNA precipitation. Excess salt can be removed br washing the RNa pellet with 70% EtoH(ACS grade). To the RNA pellet, add about 0.3 ml of room temperature (or -20oC 70% ethanol per 1.5ml tube or approximately 2 to 3 ml per 15 to 40ml tube Vortex the tube for 30 seconds to several minutes to dislodge the pellet and wash it thoroughly. Recover the RNA with a low speed spin, (approximately 3000 x g; approximately SS34 rotor), for 5 to 10 minutes at room temperature or ayos 7500rpm in a microcentrifuge, or approximately 5500rpm in Table 8. Spin Requirements for Phenol Chloroform Extractions Tube Speed Spin Time 1.5ml 10,000×g 2.0ml 15 ml 12,000×g 50ml 12,000×g RNA Purification 205

It is not difficult to find researchers who swear by GITC— phenol procedures because good-quality RNA, free from geno￾mic DNA contamination is quickly produced. However, a se￾cond camp of researchers avoid these same procedures because they often contain contaminating genomic DNA (Lewis, 1997; S. Herzer, personal communication). There is no single expla￾nation for these polarized opinions, but the following should be considered. Problems can occur in the procedure during the phenol/chloro￾form extraction step. The mixture must be spun with sufficient force to ensure adequate separation of the organic and aqueous layers; this will depend on the rotor as can be seen in Table 8.1. For best results the centrifuge brake should not be applied, nor should it be applied to gentler settings. The interface between the aqueous and organic layers is another potential source of genomic contamination. To get high￾purity RNA, avoid the white interface (can also appear cream colored or brownish) between the two layers; leave some of the aqueous layer with the organic layer. If RNA yield is crucial, you’ll probably want as much of the aqueous layer as possible, again leaving the white interface. In either case you can repeat the organic extraction until no white interface is seen. Residual salt from the precipitation step, appearing as a huge white pellet, can interfere with subsequent reactions. Excessive salt should be suspected when a very large white pellet is obtained from an RNA precipitation. Excess salt can be removed by washing the RNA pellet with 70% EtOH (ACS grade). To the RNA pellet, add about 0.3ml of room temperature (or -20°C) 70% ethanol per 1.5ml tube or approximately 2 to 3ml per 15 to 40 ml tube. Vortex the tube for 30 seconds to several minutes to dislodge the pellet and wash it thoroughly. Recover the RNA with a low speed spin, (approximately 3000 ¥ g; approximately 7500 rpm in a microcentrifuge, or approximately 5500 rpm in a SS34 rotor), for 5 to 10 minutes at room temperature or at 4°C. RNA Purification 205 Table 8.1 Spin Requirements for Phenol Chloroform Extractions Volume Tube Speed Spin Time 1.5 ml 10,000 ¥ g 5 minutes 2.0 ml 12,000 ¥ g 5 minutes 15 ml 12,000¥ g 15 minutes 50 ml 12,000¥ g 15 minutes

Remove the ethanol carefully, as the pellets may not adhere tightly to the tubes. The tubes should then be respun briefly and the residual ol removed by aspiration with a drawn out Pasteur pipet. Repeat this wash if the pellet seems unusually large Non-Phenol-Based methods Very fast, clean RNA, can process large sample numbers, pos ble genomic contamination One major drawback to using the guanidium acid-phenol nethod is the handling and disposal of phenol, a very hazardous chemical. As a result phenol-free methods, based on the ability of glass fiber filters to bind nucleic acids in the presence of chaotro- pic salts like guanidium, have gained favor. As with the other methods, the cells are first lysed in a guanidium- based buffer. The lysate is then diluted with an organic solvent such as ethanol or isopropanol and applied to a glass fiber filter or resin DNA and proteins are washed off, and the rna is eluted at the end in an aqueous buffe This technique yields total Rna of the same quality as he phenol-based methods. DNA contamination can be higher with this method than with phenol-based methods(Ambion, Inc unpublished observations). Since these are column-based proto- cols requiring no organic extractions, processing large sample numbers is fast and easy. This is also among the quickest methods for RNA isolation, usually completed in less than one hour The primary problem associated with this procedure is clogging of the glass fiber filter by thick lysates. This can be prevented by using a larger volume of lysis buffer initially. A second approach is to minimize the viscosity of the lysate by sonication(on ice, avoid power settings that generate frothing) or by drawing the lysate through an 18 gauge needle approximately 5 to 10 times. This step is more likely to be required for cells grown in culture than for lysates made from solid tissue. If you are working with a tissue that is known to be problematic (i.e, high in saccharides or fatty acids), an initial clarifying spin or extraction with an equal volume of chloroform can prevent filter-clogging problems A rea- sonable starting condition for the clarifying spin is 8 minutes at 7650xg. If a large centrifuge is not available, the lysate can be divided into microcentrifuge tubes and centrifuged at maximum speed for 5 to 10 minutes. Avoid initial clarifying spins on tissues rich in glycogen such as liver, or plants containing high molecular weight carbohydrates. If you generate a clogged filter, remove the remainder of the lysate using a pipettor, place it on top of a fresh filter, and continue with the isolation protocol using both filters. 206 Martin et al

Remove the ethanol carefully, as the pellets may not adhere tightly to the tubes. The tubes should then be respun briefly and the residual ethanol removed by aspiration with a drawn out Pasteur pipet. Repeat this wash if the pellet seems unusually large. Non-Phenol-Based Methods Very fast, clean RNA, can process large sample numbers, possi￾ble genomic contamination. One major drawback to using the guanidium acid-phenol method is the handling and disposal of phenol, a very hazardous chemical. As a result phenol-free methods, based on the ability of glass fiber filters to bind nucleic acids in the presence of chaotro￾pic salts like guanidium, have gained favor. As with the other methods, the cells are first lysed in a guanidium-based buffer. The lysate is then diluted with an organic solvent such as ethanol or isopropanol and applied to a glass fiber filter or resin. DNA and proteins are washed off, and the RNA is eluted at the end in an aqueous buffer. This technique yields total RNA of the same quality as the phenol-based methods. DNA contamination can be higher with this method than with phenol-based methods (Ambion, Inc., unpublished observations). Since these are column-based proto￾cols requiring no organic extractions, processing large sample numbers is fast and easy. This is also among the quickest methods for RNA isolation, usually completed in less than one hour. The primary problem associated with this procedure is clogging of the glass fiber filter by thick lysates. This can be prevented by using a larger volume of lysis buffer initially. A second approach is to minimize the viscosity of the lysate by sonication (on ice, avoid power settings that generate frothing) or by drawing the lysate through an 18 gauge needle approximately 5 to 10 times. This step is more likely to be required for cells grown in culture than for lysates made from solid tissue. If you are working with a tissue that is known to be problematic (i.e., high in saccharides or fatty acids), an initial clarifying spin or extraction with an equal volume of chloroform can prevent filter-clogging problems. A rea￾sonable starting condition for the clarifying spin is 8 minutes at 7650 ¥ g. If a large centrifuge is not available, the lysate can be divided into microcentrifuge tubes and centrifuged at maximum speed for 5 to 10 minutes. Avoid initial clarifying spins on tissues rich in glycogen such as liver, or plants containing high molecular￾weight carbohydrates. If you generate a clogged filter, remove the remainder of the lysate using a pipettor, place it on top of a fresh filter, and continue with the isolation protocol using both filters. 206 Martin et al